Role of caspase 1 in murine antibacterial host defenses and lethal endotoxemia

Abstract

Sepsis is thought to result from an exaggerated innate immune response to microbial components such as lipopolysaccharide (LPS), but the involvement of a specific mechanism(s) has not been identified. We studied the role of caspase 1 (Cas-1) in the murine innate immune response to infection with gram-negative bacteria and to nonlethal and lethal doses of LPS. cas-1(-/-) and Cas-1 inhibitor (Ac-YVAD-CHO)-treated cas-1(+/+) mice were two- to threefold more susceptible to lethal Escherichia coli infection than cas-1(+/+) mice. Administration of Cas-1 products, interleukin-18 (IL-18) or IL-1beta, protected three of three and six of seven mice, respectively, from lethal infection with E. coli compared to none of six of untreated mice (P = 0.0082). Therefore, cas-1 is essential for antibacterial host defense. Nonlethal (75 micro g) and lethal (500 micro g) doses of LPS induce different patterns of gamma interferon, IL-1beta, and IL-18 expression. Consequently, the role of Cas-1, which cleaves pro-IL-18 and pro-IL-1beta to their active forms, was investigated in these disparate conditions by using enzymatic assay and reverse transcription-PCR. At 75 micro g, LPS induced a transient increase in IL-1beta and IL-18 levels in serum, whereas at 500 micro g it induced a 1.5-fold-higher IL-18 level in serum, which increased till death. At 75 micro g of LPS, splenic cas-1 mRNA expression remained unchanged at all time points, but activity increased transiently at 3 h. In lethally treated mice, Cas-1 activity remained elevated until death; however, cas-1 mRNA levels increased at 3 h and decreased to basal levels by 8 h. Treatment with Cas-1 inhibitor protected mice from lethal endotoxemia. Thus, Cas-1 is essential for innate antibacterial host defenses and may represent a mechanism of innate immunity that upon excessive stimulation by microbial components may lead to endotoxic shock.

FIG. 1.

Effect of Cas-1 inhibitor on antibacterial resistance. C3H/HeN mice (five animals per group) were infected with different doses of E. coli 2 h after treatment with Cas-1 inhibitor (Ac-YVAD-CHO) at 5 mg/kg i.p. or saline and then monitored for mortality. The percent mortality was plotted against the E. coli dose, and the LD₅₀ of E. coli was determined. Symbols: ▪, without Cas-1 inhibitor; ▴, with Cas-1 inhibitor.

FIG. 2.

Cytokine levels in serum in LPS-treated C3H/HeN mice. C3H/HeN mice were treated with 75 μg (nonlethal dose [A, B, and C]) and 500 μg (lethal dose [D, E, and F]) of LPS i.p., and the IFN-γ (A and D), IL-18 (B and E), and IL-1β (C and F) levels in serum were determined at 3, 8, and 24 h after LPS treatment (75 μg) and 3, 8, and 20 h after LPS treatment (500 μg) from freshly drawn blood (P < 0.0001 by two-way ANOVA analysis). Data represent the means ± the standard errors (SE) of three mice per time point. Mice treated with 500 μg of LPS usually died between 20 and 24 h; therefore, cytokine levels in serum were determined at 20 h and not at 24 h (B, D, and F).

FIG. 3.

Expression of IL-18 mRNA and protein in LPS-treated mice. Total RNA prepared from spleen and liver tissue isolated from C3H/HeN mice before and at 3 and 8 h after administration of 75 or 500 μg of LPS was analyzed by RT-PCR for IL-18 and GAPDH mRNA expression. The levels of IL-18 mRNA in the livers (A) and spleens (B) of mice treated with 75 μg (□) and 500 μg (▪) were plotted as a ratio of IL-18 mRNA to GAPDH mRNA at the time points tested. Each point represents the mean ± SE of results from three mice. (C) Equal amounts of protein from extracts of LPS-treated mouse spleens isolated at different time points were separated on 6 to 14% gradient SDS-polyacrylamide, transferred to Immobilon-P, and immunostained with anti-IL-18 antibody (1:500). Lane 1, zero hours; lanes 2, 3, and 4, at 3, 8, and 15 h after 75 μg of LPS treatment, respectively; and lanes 5, 6, and 7, at 3, 8, and 15 h after 500 μg of LPS treatment, respectively.

FIG. 4.

Effect of LPS on Cas-1 expression. Spleens and livers of C3H/HeN mice were isolated at 0, 3, and 8 h after treatment with 75 μg (□) and 500 μg (▪) of LPS. Cas-1 activity was determined in spleen extracts prepared by the method of Thornberry (35). Enzyme activity was calculated as follows: (maximum OD₄₀₅/microgram of protein) × 10,000. Each point represents the mean ± SE of at least two animals. (A) Reactions with enzyme preparation alone and with enzyme plus substrate plus 10 μM Cas-1 inhibitor were used as controls. Total RNA was analyzed by RT-PCR for cas-1 and GAPDH mRNA levels, and the cas-1 mRNA levels in the livers (B) and spleens (C) of mice treated with 75 μg (□) and 500 μg (▪) of LPS were plotted as the ratio of Cas-1 to GAPDH. Spleen extracts containing equal amounts of protein were separated on an SDS-7.5% polyacrylamide gel, transferred to Immobilon-P membrane, and immunostained with polyclonal anti-Cas-1 antibody (1:1,000). Blot represents two separate mice per time point per treatment group. Lanes 1 and 2, zero hour; lanes 3 and 4, 75 μg of LPS at 3 h; lanes 5 and 6, 75 μg of LPS at 8 h; lanes 7 and 8, 75 μg of LPS at 15 h; lanes 9 and 10, 500 μg of LPS at 3 h; lanes 11 and 12, 500 μg of LPS at 8 h; lanes 13 and 14, 500 μg of LPS at 15 h. (D) The figure represents a single representative Western blot containing samples from the spleens of mice treated with 75 and 500 μg of LPS.

FIG. 5.

Cas-1 inhibition protects from lethal endotoxemia. C3H/HeN mice were challenged with nonlethal (75 μg) or lethal (500 μg) doses of LPS in the presence or absence of Cas-1 inhibitor (Ac-YVAD-CHO given at 10 mg/kg i.p., either 2 h before or after LPS treatment) and observed (for mortality) for the next 24 h. ✽, P < 0.0001 (Fisher exact test).